Biomarkers, such as hormones, vitamins, metabolites, can be used for the clinical diagnosis of multiple disorders and as endogenous biomarkers in endocrinology. For example, the measurement of estrogen compounds, such as estrone and estradiol can be used to evaluate ovarian function and to evaluate excess or diminished estrogen levels in a patient. Also, measurement of thyroxine can be used to quantify thyroid function.
Requirements for the clinical diagnostic testing of endogenous biomarkers in endocrinology may include highly sensitive and specific assays, the ability to analyze small sample volumes (e.g., pediatric sample volumes can be limited to less than about 200 μL), and the ability to screen for multiple analytes to accurately diagnose a disease state, e.g., an endocrine disorder. Historically, radioimmunoassay (RIA) and enzyme-linked immunoassay (ELISA) methods have been used in such clinical diagnostic testing. Immunoassay methods (IA), such as RIA and EIA, however, may suffer from low throughput, antibody cross-reactivity, which can require extra preparation for specificity, and poor scalability. Also, the analysis of endogenous biomarkers by RIA may require multiple serial dilutions for the analysis of each individual marker, which can lead to the need to make multiple adjustments to normalize sample volumes and/or the need for multiple separate tests. Also, immunoassay tesing is not particularly conducive to the analysis of multiple biomarkers in each sample. The analysis for multiple analytes in a single assay can allow for using samples of reduced size which results in assays of increased sensitivity and efficiency per sample.
An important class of hormones are the steroid hormones, such as testosterone and estrogens. Testosterone develops and maintains the male secondary sex characteristics, and promotes growth and development of sperm. Estrogen is the term used for a group of hormones of which there are three principle forms, estrone, estradiol, and estriol.
For example, relatively small variations in estrogen levels may be clinically significant. Generally, the level of estrogen in post-menopausal women, adult males, and prepubescent children is ≦10 pg/mL. Elevated estrogen levels in children may lead to precocious puberty (and short stature). In post-menopausal women, low estrogen levels may require replacement, where as levels greater than 5 pg/mL may be prognostic for certain cancers. In adult males, elevated estrogen levels may be indicative of certain disease states (testicular cancer). In adult females, reduced or elevated levels may also be indicative of certain cancers (e.g., ovarian cancer). A level of serum estrogen of 15 pg/mL is clinically different from 10 pg/mL and thus, measurement of estrogen compounds (e.g., estradiol and estrone) requires an LLOQ of 1-5 pg/mL irrespective of sample type, patient age, gender and diet.
Another important class of hormones are the thyroid hormones. Thyroxine (T4) and triiodothyronine (T3) are examples of thyroid hormones. T4 and T3 enter cells and bind to intracellular receptors. T4 and T3 are important in regulation of a number of factors including growth and development, carbohydrate metabolism, oxygen consumption, and protein synthesis. T4 acts as a prohormone, as the bulk of T3 present in blood is produced by monodeiodination of T4 by intracellular enzymes. Thyroid hormone concentrations in blood are essential tests for the assessment of thyroid function.
Thus, there is a need to develop analytical techniques that can be used for the measurement of endogenous biomarkers, and for methods that provide more sensitivity and higher throughput than RIA. Until recently, however, only GC-MS or LC-MS/MS with derivatization has been successful for small sample volumes. Thus, there is a need in the art for LC-MS/MS techniques for the analysis of endogenous biomarkers for clinical diagnosis in endocrinology capable of providing detection limits at acceptable levels, without the need for the cumbersome derivatization processes.